Sulfate reducing bacteria (SRB), as the name implies, are a group of microorganisms that are capable of reducing sulfate ion to sulfide. Microbial sulfate reduction is a widely distributed process of great ecological importance, but with significant undesirable characteristics and effects. For example, biogenic sulfide generation can induce metal corrosion (“microbial induced corrosion” or MIC) of ferrous metals in the petroleum industry, as well as weakening and decomposition of concrete structures in water collection and treatment systems, and emission of noxious and toxic hydrogen sulfide gas, which has a characteristic “rotten egg” odor, from lagoons, ponds, water tanks, and other bodies of water.
One reason for the remarkable robustness of SRBs is their ability to form biofilms. It has been postulated that biofilms are organized communities with functional heterogeneity, and which respond to their environment like tissues of higher organisms. Current thinking on the formation of such biofilms includes the initial physical attachment of cells to the surface of a solid, which consume surface adsorbed materials. The attached cells then grow into micro-colonies through the production of cellular polysaccharide, while other cells become embedded and distributed throughout the so-formed matrix. Biofilms resemble a multicellular structure, with major resistance to biocidal activity.
The concept of intercellular signaling is one that, in spite of negligible evidence, has been discussed for several decades, especially in the context of mature biofilms. One factor contributing to the intercellular signaling hypothesis is the observation that the so-called “A-factor”, which is a microbial hormone, apparently controls secondary metabolism and cellular differentiation in Streptomyces griseus. Another factor is the widespread production of N-acyl homoserine lactones (N-AHLs) in Gram-negative bacteria and the resultant quorum sensing. The N-AHLs are molecules that act as diffusible chemical communication signals (bacterial pheromones), which regulate diverse physiological processes, including bioluminescence, antibiotic production, and synthesis of coenzyme virulence factors.
The most widely studied signaling molecule is N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) which is implicated in bioluminescence. Quorum sensing has been used to describe the various N-AHL regulatory systems, which couple high cell densities and hence substrate or nutrient starvation resulting in stationary phase gene activation. N-AHL activity has been reported in naturally occurring biofilms. Rapid recovery of biofilm populations of ammonia-oxidizing organisms after ammonia starvation was reported as a result of simultaneous ammonium and OHHL addition.
Quorum sensing plays a critical role in maintaining the biofilm structure and modifying it to varying environmental conditions. Disrupting quorum sensing with synthetic molecules may be a promising method for preventing the establishment of biofilms, which has immense industrial and medical applications. Biofilms are not only responsible for clogging of pipes, but also for chronic inflammatory conditions associated with cystic fibrosis, infection of medical devices, and catheters, spread of infection in hospitals, contaminating food packaging, etc.
Nitric oxide is another signaling molecule which is used across a range of biological systems and can be used to control biofilm development and disintegration. Denitrification and dissimilatory nitrate reduction uses NO3 and NO2 as electron acceptors for oxidation of organic compounds or reduced sulfur compounds, such as hydrogen sulfide. Denitrification is the respiratory reduction of nitrate (NO3) and nitrite (NO2) via the intermediates nitric oxide (NO) and nitrous oxide (N2O). In contrast, dissimilatory nitrate reduction to ammonium (DNRA) reduce nitrate and nitrite to the final product ammonium (NH4+). FIG. 1 indicates the various forms of nitrogen compounds and their interchanges in biological systems. There are two distinct, non-overlapping conditions under which nitrite/nitrate can biologically get reduced to nitric oxide (NO). In the aerobic portion of the biofilm, which is the portion near the biomass-water interface, denitrification can be coupled with sulfide oxidation (sulfide is the electron acceptor), which is conducted by Thiobacilus denitrificans, using the enzyme sulfur dehydrogenase, as mentioned by Simpson and Holden (Composition for Odor Control, Simpson, G. D. and G. W. Holden, Pub. No.: US 2005/0115895 A1, Pub. Date: Jun. 2, 2005).
In the anaerobic portion of the biofilm, which is near the biofilm-solid support surface interface, denitrification is effected by nitrite reductase, as shown in Table 1, below. The electron acceptor in this case is an organic compound, present in the liquid phase, such as oil, and the like. The formation of nitric oxide in the anaerobic portion of the biofilm, which is responsible for biofilm attachment to the solid surface, can result in biofilm dispersal and subsequent removal of the entire biofilm, a mechanism that would not result in complete removal of biofilm, when occurring in the aerobic portion of the biofilm by the nitrate/nitrite reducing, sulfide oxidizing bacteria (Thiobacillus denitrificans).
Enzyme Technology.
Enzymes are biological catalysts that can be used to direct a chemical transformation. They are grouped into six functional classes and numerous subclasses by the Enzyme Commission of the International Union of Biochemists (Report of the Commission on Enzymes of the International Union of Biochemistry, Oxford: Pergamon Press: 1961; Enzyme Nomenclature: Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, New York: Academic Press: 1992), assigning each enzyme a unique four digit number.
Enzyme technology has recently received extensive interest, especially in environmental treatment using biological systems, such as bacteria, fungi, or other microorganisms. Previous researchers have used enzymes in activated sludge systems as indicators of specific microbial populations, to measure active biomass, as indicators of chemical oxygen demand, and as indicators of phosphorus level.
In recent years interest has increased in the use of specific enzymes for treatment of aqueous systems in place of live cultures. Use of living microorganisms for treatment presents several problems, which include (1) the inability of microorganisms to survive under stringent conditions, such as high temperature, low or high pH, and the like; (2) the need for nutrients and other substrates, such as oxygen, nitrogen, phosphorus, and the like for microbial growth, thereby requiring biostimulation; (3) competition from other indigenous organisms that are better adapted to the field conditions, thereby requiring bioaugmentation; (4) generation of biomass, which has to be handled as a by-product; (5) mass transfer limitations, which require mixing due to aggregation, settling, and the like; and (6) slow degradation rates, which severely limit the practicality of microbe-based treatments.
Reduction of sulfate to sulfide requires an organic electron donor molecule, e.g., lactic acid, which is used by the SRBs, such as Desulfovibrio and Desulfuromonas species, to reduce sulfate to hydrogen sulfide and concomitantly form bicarbonate, which results in an increase in pH (Equation 1). Soluble metal salts react with the sulfide ion in-situ to produce insoluble metal sulfides (Equation 2), thereby reducing the metal (M) concentrations to acceptable levels. Bicarbonate ions react with the protons to form carbon dioxide and water, thus removing acidity from the solution as carbon dioxide gas (Equation 3).3SO42−+lactate - - ->3H2S+6HCO3−  (1)H2S+M2+- - ->MS(precipitate)+2H+  (2)HCO3−+H+- - ->CO2(gas)+H2O  (3)
The above reactions reportedly have been used to remediate sulfate rich waters, such as acid mine drainage pits, thiosulfate in the photographic industries, sulfite in tanneries, and to treat wastewater from power plants, in which sulfur dioxide in the flue gases is removed using lime, resulting in waters containing sulfate.
Several enzymatic reactions are known to be involved in sulfate reduction. For example, adenosine 5′-phosphosulfate (APS), which is synthesized from sulfate and adenosine triphosphate (ATP) by the enzyme ATP sulphurylase (Enzyme classification 2.7.7, Table 1), serves as a nucleoside sulfate donor in sulfate reduction. APS is then broken down into sulfite and adenosine monophosphate (AMP) by APS reductase (Enzyme Classification 1.8.99, Table 1), followed by reduction to sulfide by sulfite reductase (Enzyme Classification 1.8.99, Table 1).
TABLE 1Listing of Enzymes Involved in Microbial Sulfate Reduction.No.(Reference)Classification (Properties)Reaction1.7.2.2Nitrite ReductaseR—NO2 -------> R—H AerobicDonors: Nitro compounds3NAD(P)H -------> 3NADAcceptors: cytochrome orR—NO2 -------> R—NH2 Anaerobiccopper3NAD(P)H -------> 3NAD1.13.11.18Sulfur dioxygenaseMSn -----> M++ + S2− ----> S8 ---> SO42−MSn ------> S2O3− -----> SO42−Fe2+ ---> Fe3+1.8.99Sulfite reductasesSO42− -----> SO32−SO32− -----> HS−1.1.1OxidoreductasesR—C—OH ------> R—CO3−NADH -----> NAD2.7.7Transfers phosphate to OH;CH2OH ---------> CH2OPDonor: ATP; Acceptor: OHATP -------> ADPInhibition of Sulfate Reduction.
Inhibition of biogenic sulfide production is typically attempted using one or more of the following approaches: (1) application of biocides; (2) use of nitrate; and (3) use of nitrite.
Biocides that reportedly have been used for inhibiting sulfide-producing bacteria include benzalkonium chloride, glutaraldehyde, formaldehyde, cocodiamine (1-(C6-C18)alkyl-1,3 propane diamine acetate), nitrite salts, and molybdate salts. Their reported mechanisms of action are summarized in Table 2. As the treatment level data in Table 2 indicate, very high levels of these biocides are required to inhibit hydrogen sulfide production (e.g., 50 to 500 parts-per-thousand), making these treatments expensive and environmentally undesirable.
TABLE 2Biocides for Inhibiting Sulfide-Producing Bacteria.MinimumConcentration thatChemical Nature andprevents sulfideBiocideMechanism of actionproductionBenzalkoniumQuarternary ammonium cationic 50 mg/Lchloridesurfactant; Solubilizes cellmembranes, allowing uptake ofother antimicrobialsGlutaraldehydeAldehyde; Crosslinks amino and500 mg/Lsulfhydryl groups of proteinsFormaldehydeAldehyde; Cross links amino180 mg/Lgroups of proteinsCocodiamineCationic surfactant at low pH; actssimilarly to benzalkonium chlorideNitriteSulfite analog; inhibitory of sulfite230 mg/Lreductase enzymesMolybdateSulfate analog; depletes ATP120 mg/Lreserves